Abstract

We analyzed the ratio of L:M cone photopigment mRNA in the retinas of Old World monkeys, using the method of rapid polymerase chain reaction–single-strand conformation polymorphism. The L:M cone pigment mRNA ratio in whole retina ranged from 0.6 to 7.0, with a mean of ∼1.6 (standard deviation, ±0.56; n=26). There was no change in this ratio with eccentricity up to 9 mm (∼45°), though the ratio was ∼30% greater in temporal than in nasal retina. The mRNA ratios are in good agreement with the L:M cone ratio in these same retinas, inferred from electrophysiological recordings of cone signal gain in horizontal cell interneurons. The correlation between mRNA ratios and physiological cone gain ratio supports the conclusion that both measures reflect the relative number of L and M cones.

Figures (5)

Strategy for amplification of the L and M exon-4–exon-5 coding region. A, Diagram of the X-chromosome-linked L–M visual pigment gene array. The numbered rectangles represent exons, and the solid lines connecting them represent introns. Diagram is not to scale. B, Sequence of the (top) M and (bottom) L segments that encompass the 3′ end of exon 4 and the 5′ end of exon 5, amplified with primers 148 and 78 (indicated by arrows). The dashes indicate identity in sequence between the L and the M segments. The position of intron 4 is indicated, but, because cDNA was used as the template, only exonic sequences are amplified.

Standard curve for quantification of the L:M mRNA ratio. A, Phosphor image of a single-strand conformation gel, showing the separation of L from M pigment cDNA strands. Known ratios (indicated above the lanes as input L:M mRNA ratios) of purified L and M pigment cDNA were generated and were used as templates in the competitive PCR amplification reaction by use of primers 148 (end labeled 32P) and 78 (see Fig. 1B), the sequence of which matches both L and M templates. The L- and M-derived PCR products were resolved by electrophoresis, as described in Section 2. Band densities were determined with a phophorimager. B, Plot of input and observed [±1 standard deviation (SD)] L:M cDNA ratios.

Relation of retinal location to L:M mRNA ratio. A, Schematic retina illustrates how retinal tissue was sampled. We sampled circular punches ∼3 mm in diameter along the horizontal and vertical meridians, noting retinal eccentricity, in millimeters from the fovea. Diagonal lines delineate retinal quadrants, as indicated. B, Histogram of percentage of L pigment mRNA normalized to foveal values as a function of eccentricity. Data are for the retinas listed in Table 1, with the exception of monkey 88105, for which precise eccentricity information was not available. No significant change in the L:M pigment mRNA ratio as a function of retinal eccentricity out to 9 mm was present. Values are means from five retinas; error bars indicate ±1 SD. Data at 12 mm are based on one retina. C, Histogram of percentage of L pigment mRNA by retinal quadrant (N, nasal; T, temporal; S, superior; I, inferior) normalized to the temporal quadrant; error bars indicate ±1 SD. The temporal quadrant shows the highest L pigment mRNA level; nasal retina shows a consistently large reduction of ∼30% relative to the temporal quadrant (p=0.022; two-tailed t test). Data are for the 10 eyes (9 animals) listed in Tables 1 and 2.

Plot of percentage of L cone contrast gain for H1 horizontal cells against percentage of L pigment mRNA level for the same piece of retinal tissue. The two data sets are highly cor-related (correlation coefficient r=0.85; two-tailed t test; P=0.05). A line with a slope of 1 is drawn through the data points. The values for the mRNA are the means of three measurements; error bars indicate ±1 SD. Values for the H1 cell contrast gains are taken from cells located within a 3-mm-tissue punch that was used for the mRNA analysis. Multiple measurements of M and L cone contrast gain were made on each H1 cell, and the data points are mean values. SD’s were small, ranging from 0.5% to 1.5% of the mean, and are not shown.